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Avian Pathology

ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: https://www.tandfonline.com/loi/cavp20

Protection induced by commercially available live-attenuated and recombinant viral vector vaccinesagainst infectious laryngotracheitis virus in broilerchickens

Ariel Vagnozzi , Guillermo Zavala , Sylva M. Riblet , Alice Mundt &Maricarmen García

To cite this article: Ariel Vagnozzi , Guillermo Zavala , Sylva M. Riblet , Alice Mundt & MaricarmenGarcía (2012) Protection induced by commercially available live-attenuated and recombinant viralvector vaccines against infectious laryngotracheitis virus in broiler chickens, Avian Pathology, 41:1,21-31, DOI: 10.1080/03079457.2011.631983

To link to this article: https://doi.org/10.1080/03079457.2011.631983

Accepted author version posted online: 17Oct 2011.Published online: 16 Feb 2012.

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Protection induced by commercially available live-attenuated and recombinant viral vector vaccines againstinfectious laryngotracheitis virus in broiler chickens

Ariel Vagnozzi1,2, Guillermo Zavala1, Sylva M. Riblet1, Alice Mundt1 andMaricarmen Garcıa1*

1Department of Population Health, College of Veterinary Medicine, Poultry Diagnostic and Research Center, University ofGeorgia, Athens GA 30602, USA, and 2Instituto Nacional de Tecnologıa Agropecuaria, Estacion ExperimentalAgropecuaria, Concepcion del Uruguay, 3260 Entre Rıos, Argentina

Viral vector vaccines using fowl poxvirus (FPV) and herpesvirus of turkey (HVT) as vectors and carryinginfectious laryngotracheitis virus (ILTV) genes are commercially available to the poultry industry in theUSA. Different sectors of the broiler industry have used these vaccines in ovo or subcutaneously, achievingvariable results. The objective of the present study was to determine the efficacy of protection induced byviral vector vaccines as compared with live-attenuated ILTV vaccines. The HVT-LT vaccine was moreeffective than the FPV-LT vaccine in mitigating the disease and reducing levels of challenge virus whenapplied in ovo or subcutaneously, particularly when the challenge was performed at 57 days rather than35 days of age. While the FPV-LT vaccine mitigated clinical signs more effectively when administeredsubcutaneously than in ovo, it did not reduce the concentration of challenge virus in the trachea by eitherapplication route. Detection of antibodies against ILTV glycoproteins expressed by the viral vectors was auseful criterion to assess the immunogenicity of the vectors. The presence of glycoprotein I antibodiesdetected pre-challenge and post challenge in chickens vaccinated with HVT-LT indicated that the vaccineinduced a robust antibody response, which was paralleled by significant reduction of clinical signs. Thechicken embryo origin vaccine provided optimal protection by significantly mitigating the disease andreducing the challenge virus in chickens vaccinated via eye drop. The viral vector vaccines, applied in ovo andsubcutaneously, provided partial protection, reducing to some degree clinical signs, and challenge VIRUSreplication in the trachea.

Introduction

Infectious laryngotracheitis virus (ILTV) is a member of

the genus Iltovirus, family Herpesviridae, subfamily

Alphaherpesvirinae. ILTV is taxonomically classified as

Gallid herpesvirus 1 (Davison et al., 2009), and is the

causative agent of the respiratory disease of infectious

laryngotracheitis (ILT) in chickens. The disease occurs

frequently in densely populated poultry production areas

and generates severe production losses due to at least

increased mortality, decreased egg production, delayed

body weight gain, and predisposition to other respira-

tory pathogens (Guy & Garcıa, 2008). The control of the

disease is based on vaccination and biosecurity. Two

types of live-attenuated vaccines have been utilized to

control infectious laryngotracheitis: vaccines attenuated

by multiple passages in embryonated eggs or of chicken

embryo origin (CEO) (Samberg et al., 1971); and a

vaccine attenuated by multiple passages in tissue culture

or of tissue culture origin (TCO) (Gelenczei et al., 1965).

These vaccines have been proven to be effective, parti-

cularly the CEO vaccine, producing good protection

against the disease (Fulton et al., 2000; Han et al., 2003;

Rodrıguez et al., 2008). However, live-attenuated ILTV

vaccines, in particular that of CEO, regain virulence

after consecutive passages in chickens (Guy et al., 1991).

Both vaccines (CEO and TCO) can be transmitted from

vaccinated to unvaccinated birds (Gelenczei et al., 1964;

Hilbink et al., 1987; Andreasen et al., 1989; Rodrıguez-

Avila et al., 2007), establishing latent infections in

apparently healthy chickens (Andreasen et al., 1989).

There is strong evidence to relate recent ILTV epizootics

in the USA, South America and Europe to CEO-derived

strains that persisted in the field and regained virulence

(Kirkpatrick et al., 2006; Neff et al., 2008; Oldoni et al.,

2008; Chacon et al., 2009; Chin et al., 2009). This

situation has encouraged the development of a new

generation of ILT vaccines consisting of viral vector

vaccines containing fowl poxvirus (FPV) and herpes-

virus of turkey (HVT) as vectors. The HVT-LT

vaccine carries the US-6 and US-7 ILTV genes encoding

viral glycoproteins D and I (gI), respectively (Hein,

2008); and the FPV-LT vaccine carries the UL-27 and

UL-34 ILTV genes encoding viral glycoprotein B (gB)

* To whom correspondence should be addressed:. Tel: �1 7065425656. Fax: �1 7065425630. E-mail: mcgarcia@uga.edu

Avian Pathology (February 2012) 41(1), 21�31

Received 23 July 2011

ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/12/010021-11 # 2012 Houghton Trust Ltdhttp://dx.doi.org/10.1080/03079457.2011.631983

and membrane-associated protein, respectively (Davisonet al., 2006). Compared with CEO and TCO vaccines,the advantages of these vaccines include lack of trans-mission from bird to bird, lack of ILTV latent infectionsafter vaccination, and no reversion to virulence. Theviral vector vaccines offer a safer vaccination alternativeagainst ILTV and have been commercially available inthe USA for approximately five years. Although theywere originally licensed for subcutaneous (HVT-LT) andtranscutaneous (FPV-LT) application, the growing trendof vaccinating against Marek’s disease virus by the in ovoroute and the increased incidence of ILT eporniticsprompted the industry to use the ILTV vector vaccinesvia the in ovo route. The protection induced by HVT-LTand FPV-LT viral vector vaccines to broilers whenapplied in ovo with a non-commercial one-needle vacci-nator indicated that these vaccines slightly reducedclinical signs when applied in ovo but did not reducesignificantly the replication of ILTV challenge virus inthe trachea (Johnson et al., 2010). The objectives ofthe current study included: to evaluate the protectioninduced by ILTV viral vector vaccines when appliedin ovo using a commercially available in ovo vaccinationsystem or subcutaneously at hatch. To compare theprotection efficacy of viral vector vaccines with that ofthe live attenuated vaccines applied via eye-drop. Protec-tion was defined as the ability to prevent clinical signs,reduce replication of the challenge virus in the trachea,and maintain body weight gain after challenge. Inaddition, serum antibody responses elicited by viralvector vaccines pre-challenge and post challenge wereevaluated.

Materials and Methods

ILTV vaccines and challenge isolate. The vaccines used in the present

study were the viral vector HVT-LT vaccine Innovax ILT†

(Intervet/

Schering-Plough Animal Health, Millsboro, Delaware, USA), the viral

vector FPV-LT vaccine Vectormune†

FP-LT (Ceva, Lenexa, Kansas,

USA), the serotype 3 HVT vaccine (Merial Inc., Gainsville, Georgia,

USA), the CEO vaccine Trachivax†

(Intervet/Schering Plough Animal

Health), and the TCO vaccine LT-IVAX†

(Intervet/Schering Plough

Animal Health). The vaccines were prepared as recommended by their

respective manufacturers and titrated after their delivery to confirm the

dose applied. The CEO and TCO vaccines and the virulent field isolate

63140 (Vagnozzi et al., 2010) were titrated in chicken kidney (CK) cells

as previously described (Rodrıguez-Avila et al., 2007). Titres in CK cells

were expressed as the 50% tissue culture infectious dose (TCID50) based

on cytopathic effect and estimated by the Reed and Muench method

(Reed & Muench, 1938). The HVT-LT, HVT, and FPV-LT were titrated

in secondary chicken embryo fibroblasts. The FPV-LT titration was

performed in 24-well plates and titres were expressed as TCID50 using

the Reed and Muench method. The HVT-LT and HVT vaccines were

titrated in 60 mm dishes, where plaques were counted as previously

described (Wakenell et al., 2008) and titres were expressed as plaque

forming units (PFU) per millilitre.

Experimental design. The protection induced by ILTV viral vector

vaccines in broilers when challenged at 35 and 57 days of age was

compared with live-attenuated vaccines. In the present study,

live-attenuated ILT vaccines were administered as a full dose, and the

recombinant vectored vaccines were administered as a half dose to

mimic broiler industry practices. The viral vector vaccines were

administered in ovo and subcutaneously at hatch while the live-

attenuated vaccines, CEO and TCO, were administered via eye drop

at 14 days of age. The experimental design and treatments utilized in

this study are summarized in Table 1. Three hundred broilers’ eggs from

a local commercial source were incubated in a small-scale hatcher

(Natureform Inc., Jacksonville, Florida, USA) from 0 to 21 days. At 18

days of embryo age, the eggs were candled, infertile eggs were removed,

and the fertile embryos were divided into four groups, of which two were

vaccinated in ovo using a commercial Inovoject†

vaccinator (Pfizer

Animal Health, Durham, North Carolina, USA). One group of 40

embryonated eggs was vaccinated with half a dose of HVT-LT vector

vaccine (Innovax ILT†

; Intervet/Schering-Plough Animal Health); a

second group of 40 embryonated eggs was vaccinated with half a dose of

the FPV-LT vector vaccine (Vectormune†

FP-LT; Ceva); a third group

of 40 embryonated eggs were vaccinated with a full dose of HVT vaccine

(Merial Inc.); and 120 embryonated eggs remained unvaccinated. At

hatch, 90 of the non-vaccinated chicks were separated into three groups:

one group of 30 chicks was vaccinated subcutaneously with half a dose

of the HVT-LT vaccine; 30 chicks were vaccinated with half a dose of

the FPV-LT vaccine; and the remaining 30 chicks were vaccinated

subcutaneously with a full dose of the HVT serotype 3 vaccine. All

vaccinated and non-vaccinated chickens were identified by wing tags

and housed in separated floor pens located at the Poultry Diagnostic

and Research Center (Athens, Georgia, USA). At 14 days of age, 24

non-vaccinated hatch-mates were transferred to four polycarbonate

Plexiglas units with filtered-air, negative-pressure isolation units located

at the Poultry Diagnostic and Research Center, and fed a standard diet

and water ad libitum. Chickens were distributed in four units, six

chickens per unit, 12 were vaccinated with the Trachivax†

(CEO)

vaccine (Intervet/Schering Plough Animal Health), and 12 chickens

were vaccinated with the LT-IVAX†

(TCO) vaccine (Intervet/Schering

Table 1. Experimental design and treatments.

Vaccination Challenge

Treatment In ovo Subcutaneous Eye drop 35 days of age 57 days of age

NVx-NCh � � � � �CEO � � 14 DO IT/EDa ND

TCO � � 14 DO IT/EDa ND

HVT-LT io 18 DOE � � IT/EDa IT/EDb

HVT-LT sc � 1 DOA � IT/EDa IT/EDb

HVT io 18 DOE � � IT/EDa ND

HVT sc � 1 DOA � IT/EDa ND

FPV-LT io 18 DOE � � IT/EDa IT/EDb

FPV-LT sc � 1 DOA � IT/EDa IT/EDb

NVx-Ch � � � IT/EDa IT/EDb

DOE, days old embryo; DOA, days of age; ND, not done. aIT/ED, intratracheally/eye drop at a dose of 1600 TCID50/chicken.bIntratracheally/eye-drop at dose 1000 TCID50/chicken.

22 A. Vagnozzi et al.

Plough Animal Health). Both live-attenuated vaccines were adminis-

tered at a full dose via eye drop. At 34 days of age, 11 or 12 chickens

from the in-ovo-vaccinated groups, 11 or 12 chickens from the

subcutaneous-vaccinated groups, and 22 to 24 non-vaccinated chickens

were transferred to 16 filtered-air and negative-pressure Plexiglas

isolation units located in the same room where the CEO and TCO

vaccinated chickens were housed. The in ovo, subcutaneously vaccinated

and non-vaccinated chickens were distributed into two isolation units

per group, 5 or 6 chickens per unit, identified as: non-vaccinated, non-

challenged group (NVx-NCh), HVT-LT in ovo vaccinated challenged

group (HVT-LT io), HVT-LT subcutaneously vaccinated challenged

group (HVT-LT sc), HVT in ovo vaccinated challenged group (HVT io),

HVT subcutaneously vaccinated challenged group (HVT sc), FPV-LT in

ovo vaccinated challenged group (FPV-LT io), FPV-LT subcutaneously

vaccinated challenged group (FPV-LT sc), non-vaccinated challenged

group (NVX-Ch), CEO vaccinated challenged group (CEO), and TCO

vaccinated challenged group (TCO). All chickens were weighed before

challenge. With the exception of the NVx-NCh group, all chickens were

challenged at 35 days of age with the virulent ILTV field isolate 63140

administered in a total volume of 200 ml, 100 ml intratracheally and 50 ml

via eye drop per eye. Chickens in the NVx-NCh group were mock-

inoculated with phosphate-buffered saline (PBS) solution instead. At 56

days of age a second group of chickens from NVx-NCh, HVT-LT io,

HVT-LT sc, FPV-LT io, FPV-LT sc and NVX-Ch were weighed and

transferred to 12 isolation units, two units per group, 5 or 6 chickens per

unit, and were provided with feed and water ad libitum. With the

exception of the NVx-NCh group of chickens, at 57 days of age all

chickens were challenged with the virulent ILTV field isolate 63140 as

described above.

Protection was evaluated by scoring clinical signs daily between 3

and 7 days post challenge (d.p.c.). The challenge virus present in the

trachea was quantified by real-time polymerase chain reaction (PCR) at

3, 5 and 7 days d.p.c. Blood samples were collected before challenge and

at 7 d.p.c. for antibody detection. All chickens were humanely

euthanized at 7 d.p.c.

Clinical signs and body weight. Clinical signs were recorded daily from 3

to 7 d.p.c. as previously described (Vagnozzi et al., 2010). Briefly,

breathing patterns, conjunctivitis, and the level of depression were

scored on a scale of 0 to 3: normal (0), mild (1), moderate (2) and severe

(3). Mortality was given a total score of 9. The median clinical sign

score per group per day was calculated, and differences among groups

were analysed statistically. All chickens were weighed at 34 days of age

(pre-challenge) and at 42 days of age (7 d.p.c.). The body weight gained

per bird was calculated using the following formula:

Final weight FWð Þ � inital weight IWð Þ=FW:

The average weight gained for chickens challenged at 35 days of age was

calculated per group. Body weight gained after challenge at 57 days of

age was not recorded.

DNA extraction from trachea. Tracheal swabs were placed in 1 ml sterile

PBS solution containing 2% antibiotic-antimycotic 100� (Gibco,

Grand Island, New York, USA), and 2% newborn calf serum (Gibco).

All samples were stored at�808C until processing. DNA extraction from

tracheal swabs was performed using the MagaZorb†

DNA mini-prep

96-well kit (Promega, Madison, Wisconsin, USA), following the

manufacturer’s recommendations with some modifications. Briefly, 70

ml swab suspension or supernatant from feather pulp samples was

harvested after centrifugation and incubated with 7 ml Proteinase K and

50 ml lysis buffer at 568C for 10 min in a 96-well plate; 20 ml magnetic

beads was added along with 125 ml binding buffer to each well and

incubated for 10 min at room temperature. The supernatant was

separated and the beads were washed twice with washing buffer. Finally,

the DNA was eluted from the beads with 100 ml elution buffer.

Quantitative real-time PCR for ILTV in tracheal swabs.. ILTV DNA

from tracheal swabs was quantified by real-time PCR in a duplex assay

normalized for the host DNA as previously described (Vagnozzi et al.,

2010). Briefly, the viral genome load for each sample was normalized to

the amount of the host DNA (Niesters et al., 2001). The relative amount

of the viral DNA was calculated as the log10 of 2�DDCt:

DDCtt � DCtbt;

where DCtt is the amount of the targeted ILTV gene normalized against

the amount of the collagen gene (internal control gene) from the NVx-

Ch and Vx-Ch groups of birds, and DCtbt represents the amount of the

targeted ILTV gene normalized against the amount of the collagen gene

detected before challenge (Livak et al., 2001).

Indirect immunofluorescent antibodies. To determine antibody titres

specific to the ILTV antigens expressed by the viral vector vaccines,

serum samples collected pre-challenge and 7 d.p.c. were tested with an

indirect immunofluorescent antibody (IFA) assay using recombinant gB

and gI proteins as antigens. Briefly, the coding sequences of gI,

expressed by the HVT-LT vaccine, and gB, expressed by the FPV-LT

vaccine, were amplified by PCR using high-fidelity Pfx poly-

merase (Invitrogen, Carlsbad, California, USA) with primers specifying

unique restriction sites for cloning in the eukaryotic expression vector

pcDNA3 (Invitrogen). The 3?-end primers of both glycoprotein genes

contained a coding sequence for an RGS-6xHis-tag for detection with a

commercially available monoclonal antibody (Qiagen, Hilden,

Germany). The gB coding sequence was amplified using primers

5 ?-ACG GGATCC ATGCAATCCTACATCGCCGTG-3 ?and 5 ?-CGTGCGGCCGCCTAATGGTGATGGTGATGGT-

GACTTCCTCTTTCGTCTTCGCTTTCTTCTGCC-3 ?, and

the gI coding sequence was amplified using primers 5 ?- AC G

G G AT C C ATG G C AT C G C TAC T TG G A AC T C - 3 ? and 5?-CGTGCGGCCGCCTAATGGTGATGGTGATGGTGACTTCCTCT-

CATTTTTATTGAGTCGGGCGAGC-3?. Recombinant plasmids were

sequenced to confirm accuracy of the inserts. Expression of the

recombinant proteins was initially assayed by IFA after transient

transfection of LMH cells using a monoclonal antibody to RGS-

6xHis (Qiagen) and convalescent sera from ILTV-infected chickens.

LMH cells were seeded in 96-well plates and transfected using Mirus

TransIT-LT1 transfection reagent (Roche, Madison, WI) at 60 to 80%

confluency. At 48 h post transfection, cells were fixed with cold ethanol

for 30 min at room temperature (RT) and air-dried. Sera were diluted in

PBS at a 1:200 dilution and incubated with the fixed cells in a humid

chamber for 1 h at RT. After incubation, cells were washed with PBS

three times for 5 min. FITC-conjugated anti-species antibodies (Sigma-

Aldrich, St. Louis, MO) were diluted in PBS containing 0.001% Evan’s

blue at a 1:200 dilution and incubated for 1 h at RT. Cells were washed

with PBS three times for 5 min, briefly rinsed with distilled water, air-

dried and mounted in 2.5% 1,4-diazabicyclo-[2.2.2]-octane in 90%

glycerol. Plates were examined by conventional fluorescence microscopy

using an Axiovert 40 CFL fluorescence microscope (Zeiss, Thornwood,

New York, USA). Sera collected pre-challenge and at 7 d.p.c. from the

NVx-NCh, CEO, TCO, HVT-LT io, HVT-LT sc, HVT io, HVT sc,

FPV-LT io, FPV-LT sc, and NVx-Ch groups were tested by both gB and

gI IFA. Similarly, sera were collected at 7 d.p.c. from NVx-NCh, HVT-

LT io, HVT-LT sc, FPV-LT io, FPV-LT sc, and NVx-Ch groups of

chickens challenged at 57 days of age were tested by gB and gI IFA.

Serum samples with titres ]1:200 were considered positive and the

percentage of positive samples per group was reported.

Serology. Serum samples were analysed by commercial enzyme-linked

immunosorbent assays (ELISAs) using the LT ELISA kit (ProFLOCK†

LT ELISA Kit; Synbiotics Corp., San Diego, California, USA)

following the manufacturer’s instructions. ILTV neutralizing antibody

titres were also determined by virus neutralization (VN) assay

performed in 96-well plates with CK cells as previously described

(Thayer et al., 2008). Briefly, each serum sample was heated at 568C for

30 min to destroy heat-sensitive, non-specific virus inhibitory sub-

stances. Twelve two-fold serial dilutions were performed for each serum

in minimum essential medium with 2% antibiotic-antimycotic solution

100� (Gibco) and 2% newborn calf serum (Gibco). Each dilution was

mixed with 100 TCID50 of the ILTV 63140 isolate in a 96-well plate and

incubated for 60 min at 378C. After incubation the serum�virus mix was

transferred and absorbed into CK cell monolayers. The plate was

incubated for 5 days at 398C. After 5 days of incubation, the plate was

examined for the presence of ILTV cytopathic effect and the VN titre

Vaccines against infectious laryngotracheitis 23

was estimated as the reciprocal of the first serum dilution were ILTV

cytopathic effect was observed. The geometric mean titre for ELISA

and VN was determined in serum samples for each treatment group.

ELISA and VN assays were performed in serum samples collected

pre-challenge (34 days of age), at 7 d.p.c. from NVx-NCh, CEO, TCO,

HVT-LT io, HVT-LT sc, HVT io, HVT sc, FPV-LT io, FPV-LT sc, and

NVx-Ch groups of chickens when challenged at 35 days of age, and at 7

d.p.c. from NVx-NCh, HVT-LT io, HVT-LT sc, FPV-LT io, FPV-LT sc,

and NVx-Ch groups of chickens when challenged at 57 days of age.

Statistical analysis. Data were entered into an Excel spreadsheet

(Microsoft†

Office Excel† 2007) and analysed with SPSS Statistics

17.0 software (https://www.spss.com; SPSS-IBM Inc., Chicago, Illinois,

USA). For clinical sign score analysis, the Kruskal�Wallis test was

independently used to compare the median clinical sign scores for each

day post challenge, and multiple pair-wise comparisons were performed

for post-hoc comparisons. Normalized challenge viral loads [log10

(2�0^Ct)] for each time point were compared independently among

groups using one-way analysis of variance. When significant differences

were found at the 5% level of significance, Bonferroni’s method for

multiple pair-wise comparisons was used to detect differences between

pairs. The average weight gain was also evaluated using the analysis of

variance method.

Results

Vaccines and challenge virus titres. Once the vaccineswere reconstituted as recommended by the manufac-turers and administered in ovo, subcutaneously and viaeye drop, titres of the working dilutions were deter-mined. The Innovax ILT

†

HVT-LT vaccine titre was7460 PFU/ml, the Vectormune

†

FP-LT vaccine titre was6.30 x 103.0 TCID50/ml, the HVT vaccine titre was 9000PFU/ml and 373, 315, and 450 PFU of each vaccine

were administered, respectively, in ovo and subcuta-neously. The Trachivax

†

CEO vaccine titre was1�103.63 TCID50/ml, the LT-IVAX

†

TCO vaccine titrewas 1�104.17 TCID50/ml or 200 and 790 TCID50 wereadministered via eye drop per bird, respectively. The vialstock of virulent field isolate 63140 at a titre of 3.98�104

TCID50/ml was utilized for challenge at 35 and 57 daysof age, and each chicken received approximately 8000TCID50 of the challenge virus.

Clinical signs score evaluation. Clinical signs scoreswere recorded for all groups of chickens from 3 to 7d.p.c. Throughout both challenge experiments, 3 to7 d.p.c., the predominant clinical signs observed werebreathing difficulties and depression, followed by con-junctivitis; no mortalities were observed. For bothchallenge experiments the peak of clinical signs was at5 d.p.c., and by 7 d.p.c. most chickens had cleared theconjunctivitis and only few showed signs of mildrespiratory distress and depression (data not shown).The median clinical signs scores recorded at 5 d.p.c. forchickens challenged at 35 or 57 days of age are shown inFigure 1a,b, respectively. For chickens challenged at 35days of age, clinical scores for the TCO, HVT-LT ioand HVT-LT sc groups were not significantly different(P � 0.05) to the scores recorded for the CEOvaccinated and the NVx-NCh (negative control), or theFPV-LT io and FPV-LT sc groups. However, the FPV-LTio and FPV-LT sc groups clinical scores were signifi-cantly higher (P B 0.05) than those presented by theCEO vaccinated and the NVx-NCh groups. The FPV-LTio vaccinated group of chickens clinical signs scores wereno different (P � 0.05) from the NVx-Ch (positive

Figure 1. Median clinical sign scores recorded at 5 d.p.c. with the virulent ILTV strain 63140. 1a: Clinical sign scores 5 d.p.c. in

commercial broilers vaccinated with HVT-LT, FPV-LT in ovo (io), HVT-LT, FPV-LT subcutaneously (sc) at hatch, HVT io, HVT sc at

hatch, CEO via eye drop at 2 weeks of age, TCO via eye drop at 2 weeks of age, and a non-vaccinated group (NVx-Ch) challenged at 35

days of age. 1b: Clinical sign scores 5 d.p.c. in commercial broilers vaccinated with HVT-LT, FPV-LT io, HVT-LT, FPV-LT sc and a non-

vaccinated group (NVx-Ch) challenged at 57 days of age. Significant differences among treatments are presented with different lowercase

letters.

24 A. Vagnozzi et al.

control) group scores, while the FPV-LT sc grouppresented scores significantly lower (P B 0.05) thanthose recorded for the FPV-LT io, HVT io, HVT sc andNVx-Ch groups (Figure 1a). As expected, no significantdifferences (P � 0.05) were observed among scores ofthe HVT io, HVT sc, and NVx-Ch groups. At 57 days ofage, clinical scores were recorded for four vaccinatedgroups of chickens (HVT-LT io, HVT-LT sc, FPV-LT ioand FPV-LT sc) and two non-vaccinated groups (NVx-NCh and NVx-Ch). Similarly to the chickens challengedat 35 days of age, no significant differences (P � 0.05)were observed between the HVT-LT io and HVT-LT scvaccinated groups and the NVx-NCh group. The FPV-LT io and FPV-LT sc scores were significantly higher(P B 0.05) than scores recorded for the NVx-NChgroup and were significantly lower (P B 0.05) thanscores recorded for the NVx-Ch group (Figure 1b). By 6d.p.c., no differences (P � 0.05) in clinical signs scoreswere observed for the NVx-NCh, HVT-LT io, HVT-LTsc, and FPV-LT sc groups of chickens challenged ateither 35 or 57 days of age. However, the FPV-LT iogroup still presented clinical scores significantly higher(P B 0.05) than the NVx-NCh group either for chickenschallenged at 35 or at 57 days of age. At 7 d.p.c. theclinical scores for all of the vaccinated groups werestatistically similar (P � 0.05) to the NVx-NCh group(data not shown).

ILTV loads in trachea post challenge. Viral load levels inthe trachea were quantified at 3, 5 and 7 d.p.c. byquantitative PCR and are presented in Table 2. The peakof virus replication was observed at 3 d.p.c., as indicatedby 6.84 and 6.33 log10 2�DDCt values for the NVx-Chgroups of chickens challenged at 35 and 57 days of age,respectively. At 3 d.p.c., viral loads detected in the HVT-LT io and HVT-LT sc vaccinated groups were signifi-cantly lower (P B 0.05) than those detected for theNVx-Ch groups of chickens challenged at either 35 and57 days of age. At d.p.c. the HVT-LT io and HVT-LT scgroups, challenged at 57 days of age, showed viral loadlevels that were significantly lower (P B 0.05) thanthose of the NVx-Ch group, while when challenged at 35days of age the HVT-LT io and HVT-LT sc vaccinatedgroups showed viral load levels similar (P � 0.05) tothose detected for the NVx-Ch group. Regardless of theage when the challenge was conducted, or the day post

challenge when samples were collected (3 or 5 d.p.c.),both the HVT-LT io and HVT-LT sc vaccinated groupsof chickens presented viral load levels higher (P B 0.05)than levels detected for the NVx-NCh groups. At 3 d.p.c.viral load levels detected for the HVT-LT io and HVT-LT sc groups, when challenged at 35 days of age, weresignificantly higher (P B 0.01) than those found in theTCO and CEO vaccinated groups. However, at d.p.c. nosignificant differences (P B 0.05) were detected betweenthe HVT-LT sc and the TCO vaccinated group, whilewhen compared with the CEO the HVT-LT sc grouppresented significantly higher viral load levels. Concur-rently, at d.p.c. the HVT-LT io, HVT-LT sc, FPV-LT ioand FPV-LT sc groups all had viral load levels that werenot significantly different to the NVx-Ch group. Asexpected, the viral load levels detected for the HVT ioand HVT sc vaccinated chickens were significantlyhigher (P � 0.05) than those detected for the HVT-LTvaccinated chickens (in ovo and subcutaneously) at either3 or 5 d.p.c., but similar to those of the NVx-Ch group(P�1.00). The FPV-LT io and FPV-LT sc vaccinatedgroups presented similar viral loads (P�1.00) to thoseof the NVx-Ch group at 3 and 5 d.p.c., when thechallenge occurred at either 35 or 57 days of age. Viralload levels among all groups decreased significantlybetween 3 and 5 d.p.c., and by day 7 only the FPV-LTio, FPV-LT sc and the NVx-Ch groups of chickens hadmeasurable viral loads.

Weight gain post challenge. The average body weightgained was recorded between 34 (1 day pre-challenge)and 41 (7 d.p.c.) days of age (Figure 2). Among thevaccinated groups, those that received the FPV-LT in ovoshowed the lowest weight gain (0.2 kg), which wassignificantly lower than the weight gained by the CEO(P�0.044) and the NVx-NCh (P�0.036) groups, butnot different to the weight gained by the TCO, HVT-LTio, HVT-LT sc or FPV-LT sc vaccinated groups ofchickens.

Detection of gI and gB antibodies. In order to evaluatethe antibody response to ILTV gI and gB expressed bythe HVT-LT and FPV-LT vaccines, serum samples werecollected at 34 days of age (pre-challenge), at 41 days ofage (7 d.p.c.), and at 64 days of age (7 d.p.c.) andanalysed by IFA (Table 3). A gI antibody response was

Table 2. Trachea viral load at 3, 5 and 7 d.p.c. for chickens challenged at 35 and at 57 days of age.

Challenge at 35 days of age Challenge at 57 days of age

Group 3 d.p.c. 5 d.p.c. 7 d.p.c. 3 d.p.c. 5 d.p.c. 7 d.p.c.

NVx-NCh 0.01a (90.18)A �0.27 (90.29)A �0.18 (90.24)A 0.01 (90.32)A 0.11 (90.13)A 0.04 (90.01)A

CEO �0.15 (90.24)A �0.10 (90.88)A �0.24 (90.92)A ND ND ND

TCO 2.21 (93.30)B 1.02 (92.36)B �0.38 (90.47)A ND ND ND

HVT-LT io 5.09 (91.15)C 3.41 (91.57)C 0.16 (91.30)A,B 2.68 (92.56)B 2.67 (92.01)B 0.39 (91.27)A

HVT-LT sc 5.22 (91.48)C 2.41 (91.53)B,C �0.27 (90.41)A 2.99 (91.60)B 2.49 (91.67)B 0.10 (90.73)A,B

HVT io 7.09 (91.18)D 3.63 (90.75)C 0.62 (91.60)A,B ND ND ND

HVT sc 7.31 (90.60)D 3.86 (90.80)C 1.41 (91.85)B ND ND ND

FPV-LT io 7.40 (91.01)D 3.56 (90.69)C 0.85 (91.55)A,B 5.90 (90.44)C 5.12 (90.64)C 1.57 (91.52)A

FPV-LT sc 6.34 (91.21)D 3.70 (90.99)C 0.84 (91.94)A,B 6.26 (90.65)C 4.76 (90.23)C 1.05 (91.32)A,B

NVx-Ch 6.84 (91.51)D 3.15 (91.25)C 0.56 (91.41)A,B 6.33 (90.68)C 4.82 (90.41)C 2.08 (90.47)B

aAverage of log10 2�DDCt; uppercase superscript letters in each column indicate whether the data were statistically different between

groups within the same time-point. Different letters indicate significant differences (P B 0.05). ND, not done.

Vaccines against infectious laryngotracheitis 25

detected in 75% and 92% of the samples collected pre-challenge from the HVT-LT sc and HVT-LT io vacci-nated groups, respectively; while a gI antibody responsewas detected in only 8% of the CEO and TCOvaccinated chickens. As expected, no gI antibodieswere detected in serum samples collected pre-challengefrom the FPV-LT or HVT vaccinated groups of chickensas neither vaccine expresses gI of ILTV. With theexception of one sample (8%) in the FPV-LT scvaccinated group, no gB antibodies were detected inserum samples collected pre-challenge from the CEO,TCO, HVT-LT io, HVT-LT sc, and FPV-LT io vacci-nated groups. As expected, no gB or gI antibodies weredetected in serum samples collected pre-challenge fromthe NVx-Ch group. In serum samples collected 7 d.p.c.from chickens challenged at 35 days age, 89 to 100% ofthe samples from HVT-LT io and HVT-LT sc vaccinatedgroups had detectable gI antibody titres; while 60 to 90%of the serum samples from HVT-LT sc and HVT-LT ioshowed gI-specific antibodies when challenged at 57 daysof age. Within the FPV-LT sc and FPV-LT io vaccinatedgroups, 44% of samples had gI antibody titres when thechickens were challenged at 35 days of age; while whenchallenged at 57 days of age, gI antibodies were found in30% of the samples collected from the FPV-LT iovaccinated group and no gI antibodies were detected insamples collected from the FPV-LT sc group. Antibodiesagainst gI were found in 92% and 50% of the serumsamples collected post challenge from the CEO andTCO vaccinated groups, respectively. For the NVx-Chgroup of chickens, gI antibodies were detected in 18%and 10% of the serum samples collected 7 d.p.c. inchickens challenged at 35 and 57 days of age, respec-tively. A gB antibody response was detected in 44% and89% of the serum samples collected at 7 d.p.c. from FPV-LT io and FPV-LT sc vaccinated groups, when chal-lenged at 35 days of age, respectively. When chickenswere challenged at 57 days of age, gB antibodies weredetected in 50% and 60% of serum samples collectedfrom FPV-LT io and FPV-LT sc, respectively. With theexception of one serum sample from the HVT-LT iovaccinated group, no gB antibodies were detected insamples collected post challenge from the CEO, TCO,HVT-LT sc vaccinated groups, or the NVx-Ch group,when challenged at 35 days of age. Similarly, no gBantibodies were detected in serum samples collected postchallenge from chickens challenged at 57 days of agefrom the HVT-LT io and HVT-LT sc vaccinated groupsor the NVx-Ch group.

ILTV ELISA and virus neutralizing antibody titres. TheILTV antibody response elicited at 34 days of age (pre-challenge) and at 7 d.p.c. was also measured by ELISAand VN assay. With the exception of serum samplesfrom the CEO and TCO vaccinated groups, all remain-ing serum samples collected pre-challenge were negativeby ELISA for ILTV antibodies, including samples fromchickens vaccinated with the HVT-LT and FPV-LTvector vaccines. ELISA titres in serum samples collectedat 7 d.p.c. from vaccinated groups CEO, TCO, HVT-LTio, HVT-LT sc, FPV-LT io and FPV-LT sc challenged at35 days of age showed mean titres that ranged from 1000to 2000. Serum samples from chickens challenged at57 days of age revealed mean titres ranging from 4000 to5000 for the HVT-LT vaccinated groups, and from 1000to 3000 for the FPV-LT vaccinated groups (Figure 3a).ELISA titres for serum samples collected from the NVX-Ch groups of chickens reached mean titres of 500 at 7d.p.c. ILTV neutralizing antibodies were also determinedin serum samples collected pre-challenge and postchallenge (Figure 3b). Low virus neutralizing antibodymean titres of 1.0 to 4.0 were detected in serum samplescollected pre-challenge from HVT-LT, FPV-LT, CEOand TCO vaccinated groups. Such values were notdifferent from the values obtained for the NVx-NCh(negative control) group. For chickens challenged at35 days of age, the mean virus neutralizing antibodytitres detected at 7 d.p.c. for HVT-LT io, HVT-LT sc,FPV-LT io and FPV-LT sc vaccinated groups were 3.4,9.5, 5.3, and 12.1, respectively. For chickens challengedat 57 days of age, the mean virus neutralizing antibodytitres, detected at 7 d.p.c., for HVT-LT io, HVT-LT sc,FPV-LT io and FPV-LT sc were 6.5, 32.0, 13.0, and 76,respectively. Noticeable is the high standard deviationobserved for mean VN titres of HVT-LT and FPV-LTsubcutaneously vaccinated chickens after challenge at57 days of age. On the other hand, virus neutralizingmean titres for CEO, TCO and NVx-Ch groups ofchickens, challenged either at 35 or 57 days of age,never produced titres different to the NVx-NCh group(Figure 3b).

Discussion

The objective of the present study was to evaluate theprotection induced by ILTV viral vector vaccines inbroilers when challenged at 35 and 57 days of age. Inthe present study live-attenuated ILT vaccines were

Figure 2. Average body-weight gained after challenge at 35 days of age for each treatment group. Treatments: non-vaccinated/non-

challenged (NVx-NCh), CEO vaccinated, TCO vaccinated, herpesvirus of turkey-LT in ovo vaccinated (HVT-LT io), herpesvirus of

turkeys subcutaneously vaccinated (HVT-LT sc), FPV-LT in ovo vaccinated (FPV-LT io), FPV-LT subcutaneously vaccinated (HVT-

LT sc), non-vaccinated/challenge.

26 A. Vagnozzi et al.

administered as a full dose, and the recombinantvectored vaccines were administered as a half dose tomimic broiler industry practices. The viral vectorvaccines were administered in ovo and subcutaneouslyat hatch while the live-attenuated vaccines, CEO andTCO, were administered via eye drop at 14 days of age.As previously described (Oldoni et al., 2009), the peakof clinical signs was observed at 5 d.p.c. and the peakof challenge virus replication in the trachea wasdetected at 3 d.p.c. In a recent study comparing thepathogenicity of the 63140 isolate against the USDAstandard challenge strain in broilers, the 63140 isolateshowed characteristically earlier replication in thetrachea (Garcıa et al., 2010), which may explain theearly peak of viral replication in this study. Aspreviously documented (Johnson et al., 2010), optimalprotection was achieved by the CEO vaccine when

applied via eye drop with no clinical signs recorded andno detectable challenge virus in the trachea from 3 to 7d.p.c. Chickens vaccinated with TCO or HVT-LTpresented reduced levels of clinical signs that were notstatistically different from the clinical signs observed inthe CEO vaccinated group. However, in contrast to theCEO vaccinated chickens, in which the lack of clinicalsigns was accompanied by reduction of challenge virusto levels close to the assay detection limit (Callison etal., 2007), high levels of virus were detected 3 d.p.c. inHVT-LT io and HVT-LT sc vaccinated groups chal-lenged at 35 days of age. On the other hand, HVT-LTio and HVT-LT sc vaccinated groups, when challengedat 57 days of age, had a reduction of two logarithms inviral load levels 3 d.p.c. compared with HVT-LTvaccinated groups challenged at 35 days of age,suggesting that immunity against ILT generated by

Figure 3. ILTV antibody titres determined by ELISA and VN assay on serum samples collected pre-challenge and post challenge. 3a:

ELISA ProFLOCK†

LT (Synbiotics Corp., San Diego, California, USA). Geometric mean titres (GMT) were calculated for serum

samples from each treatment group collected 1 day pre-challenge and 7 d.p.c. using the commercial ELISA. A treatment group with GMT

higher than 500 was considered positive for ILTV antibodies by ELISA. 3b: Virus neutralizing GMT were estimated for each treatment

group on serum samples collected 1 day previous to the challenge and 7 d.p.c. VN titres higher than 4.0 were considered positive.

Vaccines against infectious laryngotracheitis 27

recombinant vaccines may take more than a month todevelop. Independently of the route of administrationor challenge age, the HVT-LT vaccine significantlymitigated clinical signs, although a significant reductionin virus levels was observed in chickens challenged at57 days but not at 35 days of age.

Furthermore, the lack of protection observed in theHVT vaccinated chickens confirmed that the ILTV gIand gD glycoproteins expressed by the HVT-LT vaccinewere responsible for the protection observed. Chickensvaccinated with FPV-LT presented higher clinical scoresand viral load levels than the HVT-LT, CEO, or TCOvaccinated groups when challenged at 35 days of age.When chickens were challenged at 57 days of age bothparameters were significantly higher than those observedfor HVT-LT vaccinated chickens. Results obtained in thepresent study disagree with protection efficiency ob-served when HVT-LT and FPV-LT were administeredin ovo and subsequently challenged at 35 days of age withthe USDA strain (Johnson et al., 2010). In Johnsonet al.’s study the FPV-LT io vaccinated broiler chickenswere better protected against clinical signs than theHVT-LT io vaccinated chickens. While no reduction inviral load levels was observed for any of the viral vectorvaccinated group of chickens 5 d.p.c. We speculate thatthe differences in protection observed in both studieswere due to a combination of factors, amongst whichprobably the most relevant is the accuracy of the in ovovaccination achieved. Optimal efficiency of these vac-cines strongly relies on proper in ovo delivery, which canbe severely affected by the precision and consistency ofthe machine utilized for delivery (Williams & Hopkins,2011). In the Johnson et al. (2010) study embryonatedeggs were inoculated with a one-needle vaccinatordesigned for laboratory use that utilizes a system bywhich a single needle punches a hole in the eggshell anddelivers the vaccine, all in a single shot. In the presentstudy a commercially produced in ovo injection systemwas used, where a hole-puncher and a needle operateasynchronously and independently. We speculate that thesystem used in the Johnson et al. study may have hadlower consistency and accuracy, probably resulting ina lower number of hits and thus lower protection forthe vaccinates receiving either one of the viral vectorvaccines. Regardless of the route of vaccination or age ofchallenge in ovo or subcutaneous vaccination with FPV-LT did not reduce viral load levels in the trachea. Tonget al. (2001) evaluated the protection induced by wingweb vaccination with a viral vector rFPV-ILTVgBvaccine, and, similar to the commercial available FPV-LT vaccine, a significant reduction in clinical signs afterchallenge was observed, although challenge virus wasisolated in 25 to 40% of the vaccinated chickens. Theseresults suggest that FPV vectors may not elicit adequatelocal or cellular immunity to efficiently clear thechallenge VIRUS from the trachea. Similarly the FPVvector vaccine encoding the haemagglutinin H5 of avianinfluenza (FPV-H5) showed the lowest efficacy toprevent avian influenza virus shedding from the respira-tory tract as compared with avian influenza inactivatedvaccines (Hsu et al., 2010). Although it has not beenthoroughly investigated, another factor to consider is thepresence of FPV maternal antibodies limiting theprotection induced by the FPV-LT vaccine. Interferenceof the FPV-H5 vaccination against highly pathogenicavian influenza has been documented due to pre-existing

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28 A. Vagnozzi et al.

immunity elicited by a primary FPV vaccination(Swayne et al., 2000). The broiler breeder flock produ-cing the fertilized eggs utilized in this study wasvaccinated against fowlpox, as virtually all breederflocks in the Southern USA are; however, we did notevaluate the maternal antibody status of their progeny.Body weight gain pre-challenge and post challenge wasnot the best parameter to measure protection amonggroups of chickens challenged at 35 days of age.However, it is important to point out that the FPV-LTio vaccinated group had the lowest weight gain amongstall the vaccinated groups. In other experiments con-ducted by our group it has been observed that bodyweight gained by FPV-LT in ovo vaccinated chickens wasbest when embryos were vaccinated at 19 rather than18 days of embryo age (data not shown), and it has beendocumented that reactions to the FPV-LT vector vaccinecan occur when administered at 17.5 to 18 daysof embryo age rather than at 19 days (Williams et al.,2010).

Detection of antibodies against ILTV glycoproteinsexpressed by viral vectors was not interpreted as ameasure of protection, but rather as an indicationthat the chickens received the vaccine and a measureof the antigenicity of ILTV glycoprotein expressed bythe vector. However, the presence of gI antibodiesdetected pre-challenge and post challenge in chickensvaccinated with HVT-LT indicated that the vaccineinduced a robust antibody response, which coincidedwith a significant reduction of clinical signs. On theother hand, gB antibodies were detected only postchallenge in FPV-LT vaccinated chickens, suggestingthat a weaker immune response was elicited against gBby FPV-LT vaccination, which coincided with presenceof clinical signs and high levels of challenge virus inthe trachea. Although the exact nature of the ILTVprotective immunity has not been completely eluci-dated, it has been documented that humoral antibodyresponse by itself does not provide the necessaryimmunity to induce effective protection from disease(Fahey et al., 1983, 1984; Fahey & York, 1990; Hondaet al., 1994). The marginal role that antibodies play inthe protection against ILTV was further confirmed asno differences in ELISA titres were observed betweenthe optimally protected CEO vaccinated and poorlyprotected FPV-LT vaccinated groups of chickens; inaddition, measurable neutralizing serum antibodieswere only detected for HVT-LT and FPV-LT vaccinatedgroups of chickens while no neutralizing antibody titreswere detected in the CEO or TCO vaccinated group ofchickens. In particular, the HVT-LT vaccine elicited astrong antibody response probably associated with themitigation of the disease; however, to comprehensivelyassess the immunogenicity of the vector vaccines, thelocal and cell-mediated immunity would need to beevaluated.

Overall the HVT-LT vaccine was more effective thanthe FPV-LT vaccine in mitigating the disease andreducing levels of challenge virus when applied in ovoor subcutaneously, while the FPV-LT mitigated clinicalsigns of the disease more effectively when administeredsubcutaneously than in ovo but did not reduce the levelsof challenge virus in the trachea. The CEO vaccineprovided optimal protection by significantly mitigatingthe disease and clearing the challenge virus from thetrachea when applied at a full dose via eye drop.

Vaccine-induced protection against virus replication

and shedding is relevant in light of the fact that field

infection in broiler chickens appears to coincide with

the age range at which such birds are sent to slaughter

plants in many regions. Thus, the likelihood of ILTV

being spread mechanically during catching and trans-

portation may be increased if the vaccines used did not

prevent viral replication in the trachea and shedding

into the environment. Protection induced by viral

vector vaccines was best when the challenge was

performed at 57 days than at 35 days of age, suggesting

that the onset of immunity for viral vector vaccines

may come later than what is required to resist early

exposure in the field; the average age when broiler

outbreaks occur in the field is around 5 to 6 weeks of

age (G. Zavala, personal communication). One possible

strategy to improve the efficacy of current commercial

ILTV viral vector vaccines is to co-administer or

co-express immunomodulators that may enhance both

humoral and cell-mediated responses and consequently

may elicit an early onset of protection. Several exam-

ples on the advantages of immunomodulators have

been documented. An HVT vector expressing IL2

increased the protection induced by infectious bursal

disease virus and infectious bronchitis virus (IBV) live

attenuated vaccines when given by in ovo vaccination

(Tarpey et al., 2007). An FPV vector co-expressing IBV

S1 protein and IL-18 administered at hatch enhanced

significantly the protective efficiency against IBV

challenge (Cheng et al., 2010), and a recombinant

FPV co-expressing type I interferon and the Newcastle

disease virus HN and F proteins increased the protec-

tive efficacy of the vaccine against Newcastle disease

virus challenge when given in ovo and at hatch (Karaca

et al., 1998). Based on the results obtained in this

study and field observations, we speculate that in their

current design the ILTV viral vector vaccines can

mitigate the disease to a certain degree, but due to

their inability to reduce challenge virus replication in

the trachea, they may not contribute to reduce suffi-

ciently the circulation of virulent strains in areas of

overwhelming field challenge were they have failed to

contain outbreaks. In a recent unpublished retrospec-

tive study of 532 documented field cases associated

with vaccinal laryngotracheitis occurring in a high-

prevalence area, 44.5% of the cases corresponded to

flocks that had received a recombinant vaccine (half

dose) against ILT as the sole vaccine for protection

against the disease. Only 2.8% of the cases occurred in

flocks vaccinated with TCO vaccines; 2.6% of them

occurred in CEO vaccinated flocks; and 0.2% of the

cases were recorded in flocks vaccinated with a recom-

binant vaccine in the hatchery and a CEO vaccine in

the field. Despite this apparent failure of recombinant

vaccines to prevent outbreaks, it is important to

underline that field experience has suggested consis-

tently that losses in recombinant vaccinated and

challenged flocks are only a fraction of the production

losses commonly experienced in non-vaccinated chal-

lenged flocks. Currently it is not known how many or

what proportion of vaccinated flocks are truly at risk in

high-challenge areas, and thus further studies are

needed to assess the performance of each vaccine under

field conditions.

Vaccines against infectious laryngotracheitis 29

Acknowledgements

The present work was supported by the US Egg and

Poultry Association Harold E. Ford Foundation (Project

No. F025).

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